Crystalline 60 per cent active acetyl pepsin has 7 acetyl groups per mol of pepsin, 3 of which are readily hydrolyzed in acid at pH 0.0 or in weak alkali at pH 10.0. The tyrosine-tryptophane content of this acetylated pepsin, measured colorimetrically, is less than pepsin by three tyrosine equivalents. Hydrolysis at pH 0.0 or pH 10.0 of the 3 acetyl groups results in a concomitant increase in the number of tyrosine equivalents. In the pH 0.0 hydrolysis experiment there is also a simultaneous increase in specific activity. The phenol group of glycyl tyrosine is acetylated by ketene under the conditions used in the acetylation of pepsin and the effect of pH on the rate of acetylation is similar in the two cases. It is concluded that the acetyl groups in the 60 per cent active acetyl pepsin, which are responsible for the decrease in specific enzymatic activity, are 3 in number and are attached to 3 tyrosine phenol groups of the pepsin molecule.